title: Structural analysis and insights into the glycon specificity of the rice GH1 Os7BGlu26 β-D-mannosidase.
abstract: Rice Os7BGlu26 is a GH1 family glycoside hydrolase with a threefold higher kcat/Km value for 4-nitrophenyl β-D-mannoside (4NPMan) compared with 4-nitrophenyl β-D-glucoside (4NPGlc). To investigate its selectivity for β-D-mannoside and β-D-glucoside substrates, the structures of apo Os7BGlu26 at a resolution of 2.20 Å and of Os7BGlu26 with mannose at a resolution of 2.45 Å were elucidated from isomorphous crystals in space group P212121. The (β/α)8-barrel structure is similar to other GH1 family structures, but with a narrower active-site cleft. The Os7BGlu26 structure with D-mannose corresponds to a product complex, with β-D-mannose in the (1)S5 skew-boat conformation. Docking of the (1)S3, (1)S5, (2)SO and (3)S1 pyranose-ring conformations of 4NPMan and 4NPGlc substrates into the active site of Os7BGlu26 indicated that the lowest energies were in the (1)S5 and (1)S3 skew-boat conformations. Comparison of these docked conformers with other rice GH1 structures revealed differences in the residues interacting with the catalytic acid/base between enzymes with and without β-D-mannosidase activity. The mutation of Tyr134 to Trp in Os7BGlu26 resulted in similar kcat/Km values for 4NPMan and 4NPGlc, while mutation of Tyr134 to Phe resulted in a 37-fold higher kcat/Km for 4NPMan than 4NPGlc. Mutation of Cys182 to Thr decreased both the activity and the selectivity for β-D-mannoside. It was concluded that interactions with the catalytic acid/base play a significant role in glycon selection.
authors.affiliation: School of Biochemistry, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand.
authors.forename: Anupong
authors.initials: A
authors.lastname: Tankrathok
authors.forename: Javier
authors.initials: J
authors.lastname: Iglesias-Fernández
authors.forename: Sukanya
authors.initials: S
authors.lastname: Luang
authors.forename: Robert C
authors.initials: RC
authors.lastname: Robinson
authors.forename: Atsuo
authors.initials: A
authors.lastname: Kimura
authors.forename: Carme
authors.initials: C
authors.lastname: Rovira
authors.forename: Maria
authors.initials: M
authors.lastname: Hrmova
authors.forename: James R
authors.initials: JR
authors.lastname: Ketudat Cairns
mesh_terms: D020134:Catalytic Domain / Q000235:genetics; D018360:Crystallography, X-Ray; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics; D006031:Glycosylation; D006868:Hydrolysis; D016297:Mutagenesis, Site-Directed; D012275:Oryza / Q000201:enzymology* / Q000235:genetics; D011487:Protein Conformation; D013379:Substrate Specificity / Q000235:genetics; D044902:beta-Mannosidase / Q000737:chemistry* / Q000235:genetics
chemical_list: D006026:Glycoside Hydrolases; D044902:beta-Mannosidase
keywords: glycoside hydrolases; rice; structural analysis; β-mannosidases